Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRP). IRP modulate mRNA utilization by binding to iron responsive elements (IRE) in the 5 or 3 untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase (c-acon). The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its 4iron-4sulfur cluster, reversibly converting it between an IRE-binding protein and c-acon. IRP1 is also phosphoregulated by protein kinase C (PKC) but the mechanism by which phosphorylation post-translationally increases IRE binding activity has not been fully defined. To investigate this, serine 138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E) or aspartate (S138D), or to non-phosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impair ed i n aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild type and mutant IRP1s could be constituted to active aconitases anaerobically. However, when exposed to oxygen the four iron-four sulfur cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than observed for wild type IRP1. Low temperature EPR spectra were obtained for the oxidized reconstituted samples and for oxidized yeast extracts. Spectra typical for the three iron form of c-acon were observed for all but the glutamate mutant.